.  (1  Xovi'mlun-  28,  1908. 


U.  S.  DEPARTMENT   OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  109. 


A.  D.  MELVIN,  CHIEF  OF  BUREAU. 


OTEOLYTIC  CHANGES  IN  THE  RIPENING 
OF  CAMEMBERT  CHEESE. 


BY 

ARTHUR  W.  DOX, 

Chemist  in  Cheese  Investigations ,  Dairy  Division. 


WASHINGTON: 

GOVERNMENT   PRINTING   OFFICE. 

1908. 


Issued  November  28, 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  109. 

A.   D.   MKLVIN,   CHIKF  OF  BUREAU. 


PROTEOLYTIC  CHANGES  IN  THE  RIPENING 
OF  CAMEMBERT  CHEESE. 


BY 

ARTHUR  W.  DOX. 

Chemist  in  Cheese  Investigations,  Dairy  Division. 


WASHINGTON:     . 
GOVERNMENT    PRINTING   OFFICE. 
1908. 


THE  BUREAU  OF  ANIMAL  INDUSTRY. 

Chiff:  A.  D.  MBLVIN. 

Assistant  Chief:  A.  M.  FAHRINGTON. 

Chief  Clerk:  CHARLES  C.  CARROLL. 

Biochemic  Division:  M.  DORSET,  chief;  JAMES  A.  EMERY,  assistant  chief. 

Dairy  Division:  En.  H.  WEBSTER,  chief;  C.  B.  LANE,  assistant  cliief. 

Inspection  Division:  RICE  P.  STEDDOM,  chief;  MORRIS  WOODEN,  R.  A.  RAMSAY, 
and  ALBERT  E.  BEHNKE.  associate  chiefs. 

Pathological  Division:  JOHN  R.  MOHLER,  chief;  HENRY  J.  WASHBURN,  assist n MI 
chief. 

Quarantine  Division:  RICHARD  W.  HICKMAN,  chief. 

Zoological  Division:  B.  H.  RANSOM,  chief. 

Experiment  Station:  E.  C.  SCHROEDER,  superintendent;  W.  E.  COTTON,  assist  am. 

Animal  Husbandman:  GEORGE  M.  ROMMEL. 

Editor:  JAMES  M.  PICKENS. 

DAIRY  DIVISION. 
Chief:  Ed.  H.  Webster. 
Assistant  Chief:  C.  B.  Lane. 
Librarian:  Miss  C.  B.  Sherman. 

DAIRY    FARMING    INVESTIGATIONS. 

Assistant  in  charge,  B.  H.  Raid;  assistant,  Duncan  Stuart. 

Dairy  buildings:  J.  A.  Conover;  architect,  K.  E.  Parks;  ventilation  experiments, 
C.  R.  Potteiger. 

Herdbook  work:  Helmer  Rabild  and  William  Hart  Dexter. 

Southern  dairying:  S.  E.  Barnes,  J.  E.  Dorman,  J.  T.  Eaton,  H.  P.  Lykes,  J.  H. 
McClain,  A.  K.  Risser,  H.  R.  Welch,  and  T.  R.  Woodward. 

HAIRY   PRODUCTS   INVESTIGATIONS. 

Assistant  in  charge,  L.  A.  Rogers. 

Butter  investigations,  Albert  Lea,  Minn.,  and  Washington  D.  C.:  Chemist, 
W.  H.  Berg;  bacteriologist.  S.  H.  Ayers. 

Swiss  cheese  investigations.  Albert  Lea,  Minn.:  In  charge,  C.  F.  Doane;  assistant, 
T.  W.  Issajeff. 

Cheese  investigations,  Madison,  Wis.:  Chemist,  S.  K.  Suzuki;  bacteriologist,  Alfred 
Larson:  cheese  maker,  J.  W.  Moore. 

Cheese  investigations,  Storrs,  Conn.:  Mycologist,  Charles  Thorn;  chemist,  Arthur 
W.  Dox;  cheese  maker,  F.  R.  Thompson. 

Milk  secretion  investigations,  Columbia,  Mo.:  Chemist,  R.  II.  Shaw;  assistants, 
J.  O.  Halverson,  A.  E.  Perkins,  and  C.  C.  Payne. 

DAIRY    MANUFACTURING    INVESTIGATIONS. 

Assistant  in  charge,  B.  D.  White:  assistant,  S.  C.  Thompso 
Creamery  records,  Albert  Lea,  Minn.:  Creamery  practice,  John  L.  Sherk;  assistants 

(collaborators),  P.  W.  Noble  and  J.  D.  Burk. 
Creamery  practice  investigations:  J.  C.  Joslin,  Robert  Me  Adam,  F.  L.  Odell,  J.  C. 

Winkjer.  and  Thomas  Corneliuson. 
Market  investigations:  New  York  City,  C.  W.  Fryhofer;  Chicago,  H.  J.  Credicott; 

San  Francisco,  C.  L.  Mitchell. 

MARKET    MILK    INVESTIGATIONS. 

Assistant  chief  of  division  in  charge;  assistants,  Lee  H.  P.  Maynard,  Ivan  C.  Weld, 
and  <«eorge  M.  Whitaker. 

RENOVATED-BUTTER    INSPECTION. 

Chief  inspector,  M.  W.  Lang.  Chicago:  assistant,  Levi  Wells,  New  York. 
2 


LETTER  OF  TRANSMITTAL. 


U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY, 
Washington,  D.  C.,  September  21 ,  1908. 

SIR:  I  have  the  honor  to  transmit  herewith  a  manuscript  entitled 
"Proteolytic  Changes  in  the  Ripening  of  Camembert  Cheese,"  by 
Arthur  W.  Dox,  chemist  in  cheese  investigations,  Dairy  Division,  and 
recommend  that  it  be  published  as  Bulletin  109  of  this  Bureau.  This 
paper  deals  with  work  carried  on  at  the  Storrs  (Conn.).  Agricultural 
Experiment  Station,  by  cooperation  between  that  station  and  the 
Dairy  Division  of  this  Bureau. 

Respectfully,  A.  D.  MELVIN, 

Chief  of  Bureau. 
Hon.  JAMES  WILSON, 

Secretary  of  Agriculture. 

3 


CONTENTS. 


Page. 

Introductory 5 

Processes  in  the  ripening  of  Cameinbert  cheese 6 

Enzymes  in  the  cheese 8 

Proteolysis  of  casein 10 

Nitrogenous  constituents  of  the  cheese 11 

Hydrochloric  acid  precipitate : 12 

Coagulable  proteins 13 

Caseoses 14 

Peptones 15 

Polypeptids 15 

Diamino-acids 16 

Monoamino-acids 18 

Ammonia 21 

Absence  of  putrefac  ivc  products 21 

Summary 22 

Acknowledgments 22 

References  to  literature. . .  23 


PROTEOLYTIC  CHANGES  IN  THE  RIPENING  OF 
CAMEMBERT  CHEESE. 


INTRODUCTORY. 

Until  comparatively  recent  years  the  changes  that  take  place  in  the 
ripening  or  curing  of  the  different  varieties  of  cheese  were  but  little 
understood.  Although  the  practice  of  cheese  making  has  been  carried 
on  for  centuries,  all  of  our  knowledge  of  the  chemical  changes  involved 
in  the  ripening  process  and  the  various  factors  that  bring  about  these 
changes  has  come  to  us  within  the  past  fifty  years.  The  earliest 
record  we  have  of  any  discussion  of  this  subject  from  a  chemical  point 
of  view  was  published  only  a  century  ago.  In  this  paper  the  French 
chemist,  Chaptal, l  a  discusses  the  ripening  of  Roquefort  cheese  and 
advances  certain  theories  to  account  for  the  changes  in  appearance  and 
flavor  which  this  cheese  undergoes  during  its  sojourn  in  the  natural 
ripening  caves.  Biological  factors  were,  of  course,  not  taken  into 
account  in  Chaptal's  paper,  for  that  phase  of  the  subject  was  unknown 
until  the  time  of  Pasteur. 

No  scientific  study  of  the  subject,  however,  was  made  until  the 
latter  half  of  the  nineteenth  century.  The  attention  of  chemists  was 
then  directed  to  Roquefort  cheese  by  a  paper  published  by  Blondeau2 
in  1864.  Blondeau  analyzed  cheeses  in  different  stages  of  ripening 
and  found  that  the  fat  content  increased  from  1.85  per  cent  in  the 
fresh  cheese  to  32.31  per  cent  in  the  cheese  two  months  old.  This 
enormous  increase  in  the  fat  he  ascribed  to  a  synthesis  of  fat  from 
protein  by  the  mold.  But  a  comparison  of  his  figures  for  the  other 
constituents  of  the  cheese  as  well  as  the  fat  with  analyses  to  be  found 
hi  any  modern  book  on  dairy  products  will  show  their  utter  im- 
possibility. This  inaccurate  work  of  Blondeau,  however,  served  the 
purpose  of  directing  the  attention  of  other  investigators  to  the  sub- 
ject, and  during  the  next  few  years  the  changes  in  the  fat  content  of 
cheese  were  studied  by  Brassier,3  Sieber,4  Jacobstahl,5  and  Von  Niigeli 
and  Loew. B 

By  this  time  the  subject  of  cheese  ripening  had  begun  to  arouse  con- 
siderable interest  among  scientific  investigators,  and  their  researches 
were  extended  to  other  varieties  of  cheese.  Swiss  cheese  and  Cheddar 

"  Th»'  figun  s  refer  to  list  of  literature  at  end  of  bulletin. 


6  CHANGES   IN    RIPENING   OF   CAMEMBERT   CHEESE. 

cheese  in  particular  received  considerable  attention.  Among  those 
who  worked  with  Swiss  cheese  were  Weidmann,7  Rdse,8  Schulze,8  and 
Winterstein.9  In  our  own  country  the  Cheddar  type  of  cheese  has 
been  made  the  subject  of  thorough  chemical  investigation,  and  in  this 
connection  reference  is  made  to  the  work  of  Babcock  and  Russell10  and 
that  of  Van  Slyke  and  Hart.11  The  German  investigators  made  a  spe- 
cial study  of  the  products  of  proteolysis,  while  those  in  this  country 
studied  more  particularly  the  factors  that  cause  the  ripening. 

All  this  work,  however,  deals  with  the  "hard"  cheeses.  In  this 
class  of  cheeses  the  ripening  factors  are  the  enzymes  or  unorganized 
ferments  present  in  the  fresh  curd  and  the  bacteria  which  occur  in 
enormous  numbers  in  the  cheese.  Such  cheeses  require  several  months 
for  ripening,  for  the  enzymes  are  present  in  very  small  amount,  and  the 
bacteria  do  not  produce  rapid  proteolytic  changes.  In  contrast  to 
the  hard  cheeses  we  have  another  distinct  class  known  as  the  soft 
cheeses.  The  cheeses  belonging  to  this  class  differ  mainly  from  those 
of  the  former  class  in  that  they  contain  a  much  higher  percentage  of 
water.  This  higher  moisture  content  is  much  more  favorable  to  the 
development  of  micro-organisms,  and  the  ripening  proceeds  with 
greater  rapidity. 

PROCESSES  IN  THE  RIPENING  OF  CAMEMBERT  CHEESE. 

The  variety  of  soft  cheese  which  wre  shall  consider  in  this  paper  is 
the  Camembert  type,  a  soft  cheese  ripened  mainly  by  a  surface  growth 
of  mold.  Of  late  years  it  has  attained  considerable  importance  in  the 
cheese  market,  and  the  public  is  now  more  or  less  familiar  with  it. 
Although  it  is  still  imported  from  France  in  large  quantities,  its  man- 
ufacture has  been  undertaken  on  a  commercial  scale  in  our  own  coun- 
try with  considerable  success.  The  researches  carried  on  by  the 
Dairy  Division  of  the  Bureau  of  Animal  Industry,  in  cooperation 
with  the  Storrs  Agricultural  Experiment  Station,  with  a  view  to 
introducing  the  manufacture  of  Camembert  cheese  into  the  United 
States,  have  given  very  gratifying  results.12  For  a  description  of  the 
details  of  its  manufacture  the  reader  is  referred  to  a  bulletin  by  T.  W. 
IssajefT.14 

The  biological  factors  essential  to  the  production  of  Camembert 
cheese  are  the  lactic-acid  bacteria,  which  are  normally  present  in 
milk,  and  two  molds,  Penicillium  camemberti  Thorn13  and  Oidium 
lactis.  Other  molds  that  may  be  present  are  generally  contamina- 
tions and  often  deleterious  to. the  cheese.  The  penicillium  is  the  mold 
that  produces  the  actual  ripening  or  digestion  of  the  curd,  while  the 
oidium  seems  to  be  connected  hi  some  way  with  the  flavor  production. 
The  oidium  by  itself  or  in  conjunction  with  the  lactic-acid  bacteria 
can  not  ripen  a  cheese  more  than  a  few  millimeters  below  the  surface. 


RIPENING    OF    CAMEMBERT    CHEESE.  7 

The  ripening  of  all  cheeses  being  essentially  a  hydrolysis  of  the  para- 
casein  or  cheese  curd  through  the  agency  of  various  enzymes,  the  end 
products  or  simple  substances  from  which  the  complex  protein  mole- 
cule is  built  up  are  set  free  in  varying  amounts  during  the  course  of 
the  ripening  period.  At  the  same  time  secondary  reactions  may 
occur  which  involve  not  only  hydrolysis,  but  also  oxidation,  reduction, 
desamidation,  and  removal  of  carboxyl  groups.  The  products  of 
simple  hydrolysis  may  thus  undergo  further  changes  with  the  forma- 
tion of  substances  differing  widely  in  chemical  constitution  from  the 
substances  from  which  they  were  derived.  The  successive  steps  of 
such  reactions  are  often  difficult  to  follow,  and  it  is  sometimes  impos- 
sible to  ascertain  precisely  what  the  mother  substance  is.  In  most 
cases,  however,  the  secondary  changes  involve  but  one  step,  the 
removal  of  a  certain  radical  from  the  original  hydrolytic  product. 
These  changes  are  caused  for  the  most  part  by  bacteria,  and  in  a 
cheese  where  the  ripening  is  produced  almost  entirely  by  other  agen- 
cies they  are  of  minor  importance.  Where  they  do  occur  they  result 
not  from  the  direct  action  of  an  enzyme  acting  outside  the  cell  wall 
of  the  organism,  but  rather  from  activities  dependent  upon  the  life 
history  and  metabolism  of  the  organism.  For  this  reason  the  center 
of  a  hard  cheese  shows  the  same  flora  and  the  same  chemical  compo- 
sition as  the  portion  near  the  rind.  With  cheeses  of  the  Camembert- 
Brie  type,  however,  there  is  a  marked  difference  in  this  respect.  The 
actual  ripening  here  is  caused  by  the  proteolytic  enzyme  of  the  mold. 
This  enzyme  is  secreted  by  the  mold  growing  on  the  surface  of  the 
cheese  and  diffuses  toward  the  center,  digesting  the  curd  through 
which  it  passes  until  the  cheese  is  ripe.  The  fact  that  the  ripening 
begins  at  the  surface  and  proceeds  toward  the  center  indicates  that 
the  enzyme  is  produced  in  the  mycelium  of  the  mold.  The  progress 
of  the  ripening  is  very  easy  to  follow,  for  the  texture  and  color  of  the 
ripened  portion  are  quite  different  from  those  of  the  unripened  curd, 
and  there  is  always  a  sharp  line  of  demarcation  between  the  two. 
The  mycelium  of  the  mold  does  not  penetrate  more  than  a  few  milli- 
meters into  the  cheese,  forming  a  sort  of  rind  which  is  removed  when 
the  cheese  is  eaten. 

The  fresh  Camembert  cheese  differs  from  the  fresh  curd  of  other 
whole-milk  cheeses  mainly  in  the  greater  amount  of  whey  it  con- 
tains. This  slightly  increases  the  relative  amount  of  the  other  pro- 
teins, lactalbumin,  whey  protein,  and  lactoglobulin,  as  well  as  that 
of  the  nonnitrogenous  constituents — lactose,  citric  acid,  and  inor- 
ganic salts.  The  amount  of  butterfat  in  the  cheese  varies  merely 
with  the  richness  of  the  milk.  Paracasein,  however,  is  the  only 
protein  present  in  any  considerable  amount,  and  the  end  products  of 
the  ripe  cheese  may  be  considered  as  derived  from  it.  Moreover, 


8  CHANGES   IN    RIPENING   OF    CAMEMBKi:  I     CHEESE. 

the  other  three  proteins  mentioned  above  yield  the  same  primary 
disintegration  products,  and  a  distinction  as  to  the  origin  of  the 
latter  can  not  be  made. 

The  proteolytic  changes  which  constitute  the  ripening  of  Camem- 
bert cheese  consist,  therefore,  in  the  changes  which  this  paracasein 
undergoes  through  the  action  of  proteolytic  enzymes.  As  has  already 
been  mentioned,  the  principal  factor  is  the  enzyme  secreted  by  the 
Camembert  penicillium.  Other  enzymes  are  present,  however,  and 
a  brief  discussion  of  these  will  follow.  No  appreciable  proteolysis 
occurs  until  after  the  cheese  is  nearly  2  weeks  old.  But  in  the 
meantime  certain  changes  take  place  in  the  character  and  solubility 
of  the  curd.  These  changes  have  been  studied  by  Bosworth.15 
The}'  consist  mainly  in  the  liberation  of  paracasein  from  combination 
with  calcium,  due  to  the  formation  of  lactic  acid  by  lactic  acid  bac- 
teria. At  the  same  time  the  paracasein  is  converted  into  a  form 
completely  soluble  in  5  per  cent  salt  solution,  and  later  it  becomes 
insoluble  again.  As  these  are  not,  strictly  speaking,  proteolytic 
changes,  a  detailed  discussion  will  not  be  given  here. 

ENZYMES    IN    THE    CHEESE. 

With  the  exception  of  the  enzyme  secreted  by  the  mold  and  of  a 
smaller  variety  of  bacteria,  Camembert  contains  the  same  ferments 
that  are  present  in  other  cheeses.  Like  the  hard  cheeses,  it  contains 
the  milk  enzyme  galactase,  the  rennet  enzyme  chymosin  added  in 
the  curdling  process,  and  lactic  acid  bacteria.  In  the  case  of  the 
Cheddar  type  of  cheese  the  action  of  these  three  factors  has  been 
studied  in  detail.  Babcock  and  Russell  found  that  galactase  and 
rennet  (pepsin)  were  important  agents  in  the  ripening  of  this  variety 
of  cheese.  According  to  Van  Slyke  and  Hart,11  the  rennet  alone  is 
capable  of  ripening  a  cheese.  In  their  experiments  the  galactase 
was  first  destroyed  by  heat  and  then  chloroform  added  to  prevent 
the  development  of  bacteria,  yet  the  ripening  went  on,  though  not 
as  rapidly  as  in  the  normal  cheese,  and  the  character  of  the  chemical 
products  was  somewhat  different.  Thus  there  was  a  predominance 
of  paranuclein,  caseoses,  and  peptones,  and  an  abnormally  small 
amount  of  amino-acids.  The  entire  absence  of  ammonia  was  very 
striking.  The  function  of  the  bacteria  in  this  variety  of  cheese  has 
been  studied  by  Rogers.18  He  came  to  the  conclusion  that  the 
enzymes  produced  by  the  bacteria  were  responsible  for  most  of  the 
digestion  beyond  the  peptone  stage,  and  consequently  the  charac- 
teristic flavors. 

In  the  short  time  required  for  the  ripening  of  Camembert  cheese 
the  rennet,  galactase,  and  lactic  acid  bacteria  produce  no  appreciable 
digestion.  This  conclusion  was  reached  by  Bosworth,  and  the  expe- 
rience of  the  writer  confirms  it.  Even  the  hard  cheeses  which  are 


ENZYMES   IN    THE    CHEESE.  9 

ripened  entirely  by  these  agents  undergo  very  little  change  during 
the  first  month.  A  Camembert  cheese,  however,  should  be  ripe  at 
the  end  of  a  month,  and  at  the  same  time  should  contain  a  greater 
amount  of  primary  digestion  products.  This  ripening  must  be  due 
almost  entirely  to  the  mold  enzyme,  for  the  interior  curd,  which  has 
not  yet  been  reached  by  this  enzyme,  but  contains  all  of  the  other 
ferments,  shows  little  evidence  of  digestion.  If  the  unripened  curd 
in  the  center  of  a  Camembert  cheese  three  or  four  weeks  old  be  sub- 
jected to  chemical  analysis  it  will  be  found  that  the  paracasein  is 
scarcely  altered  except  for  the  fact  that  it  is  liberated  from  combi- 
nation with  calcium.  The  galactase  can  not  play  more  than  a  very 
subordinate  role,  as  is  shown  by  the  fact  that  the  cheese  ripens 
normally  when  made  from  milk  which  has  been  pasteurized  at  a 
temperature  sufficiently  high  to  impair  greatly,  if  not  destroy,  the 
activity  of  this  enzyme.  Likewise  the  rennet  can  not  be  of  more 
than  minor  importance  as  a  ripening  factor.  Recent  investigations 
have  shown  that  chymosin  or  rennet  enzyme  is  identical  with  pepsin. 
But,  as  will  be  seen  later,  the  ripening  of  Camembert  cheese  bears 
no  resemblance  to  a  peptic  digestion.  The  rennet  should  show  its 
greatest  activity  in  the  interior  curd,  which  is  quite  strongly  acid. 
But  owing  to  the  short  duration  of  the  ripening  period  and  the  small 
amount  of  rennet  present,  the  proteolytic  action  of  the  latter  is  prac- 
tically negligible.  Aside  from  the  hydrolysis  of  the  casein  into  para- 
casein  and  whey  protein,  its  action  is  inappreciable.  The  unripened 
curd  shows  no  evidence  of  peptic  digestion. 

The  lactic  acid  bacteria  which  constitute  nine-tenths  of  the  bac- 
terial flora  of  the  cheese  serve  the  purpose  of  converting  the  milk 
sugar  into  lactic  acid,  thus  producing  conditions  unfavorable  to  the 
development  cf  other  bacteria.  They  are  probably  responsible  for 
the  peculiar  flavor  which  is  characteristic  of  the  acid  curd  in  the 
interior  of  a  Camembert  cheese.  Their  proteolytic  action  is  other- 
wise hardly  noticeable.  Experiments  in  which  sterile  curd  was 
inoculated  with  these  organisms  show  that  the  amount  of  diffusible 
nitrogen  increases  only  very  slightly  at  the  end  of  a  month,  even  in 
the  presence  of  calcium  carbonate,  which  neutralizes  the  acid. 

We  are  safe  in  assuming,  therefore,  that  these  three  proteolytic 
factors — the  galactase,  the  rennet,  and  the  lactic  acid  bacteria — 
have  very  little  to  do  with  the  actual  ripening  of  the  cheese,  this 
being  essentially  the  work  of  the  enzyme  from  the  mold. 

As  has  already  been  pointed  out,  the  mold  of  Camembert  cheese 
(Penicillium  caniemberti)  secretes  a  powerful  proteolytic  enzyme, 
which  is  undoubtedly  the  most  potent  factor  in  the  ripening  of  this 
cheese.  The  fact  that  the  ripening  begins  at  the  surface  and  proceeds 
toward  the  center  indicates  that  the  enzyme  is  produced  in  the  myce- 
lium of  the  mold  and  diffuses  inward.  The  diffusibility  of  this  enzyme 
58056— No.  109—08 2 


10  CHANGES   IN    RIPEX1XU    OF    CA.MK.M  IJKK T    CHEESE. 

is  also  shown  by  the  fact  that  synthetic  culture  media  upon  which 
this  mold  has  grown  for  some  time  have  a  marked  proteolytic  activity. 
Experiments  are  now  being  instituted  to  determine  the  exact  nature 
of  this  enzyme  and  the  extent  to  which  it  will  hydrolyze  certain  proteins. 
The  results  thus  far  obtained  seem  to  indicate  that  it  is  of  the  nature 
of  erepsin.  It  attacks  casein  and  peptone  readily,  but  is  without 
action  upon  fibrin  and  coagulated  egg  albumin. 

Vines17  lias  shown  that  erepsin  is  very  widely  distributed  jn  the 
vegetable  kingdom.  This  vegetable  "ereptase,"  as  he  calls  it,  differs 
from  animal  erepsin  in  that  it  is  most  active  in  the  presence  of  th'e 
natural  acid  of  the  plant.  The  addition  of  other  acids,  or  of  an 
alkali,  greatly  impairs  its  activity.  As  long  as  the  acidity  is  due 
entirely  to  acid  phosphates,  the  activity  of  this  ereptase  is  very 
pronounced,  but  the  presence  of  free  acid  in  the  medium  is  inhibitory. 
It  is  readily  seen,  therefore,  that  a  fresh  Camembert  cheese  offers 
very  favorable  conditions  for  the  action  of  ereptase.  In  the  first 
place,  casein  and  paracasein  are  readily  attacked  by  this  enzyme. 
In  the  second  place,  the  lactic  acid  produced  by  the  bacteria  does  not 
accumulate  but  combines  with  the  calcium  phosphate,  forming 
calcium  lactate  and  mono-calcium  phosphate.  The  acidity  of  the 
cheese  is  due  to  the  presence  of  the  latter  salt. 

In  this  connection,  however,  it  must  be  remembered  that  the  same 
results  are  not  necessarily  obtained  with  enzymes  in  the  presence 
of  an  antiseptic  as  with  organisms  in  vivo.  In  the  case  of  Camembert 
cheese  where  the  proteolysis  is  effected  by  diffusion  of  the  enzyme 
rather  than  by  diffusion  of  the  substratum,  the  conditions  more 
nearly  approach  those  met  with  in  an  artificial  digestion  experiment. 
Nevertheless  there  are  certain  striking  differences  which  will  be 
pointed  out  later.  A  discussion  of  the  enzymes  obtained  from  a 
pure  culture  of  this  organism  will  be  reserved  for  a  future  paper. 

PROTEOLYSIS    OF    CASEIN. 

When  casein  or  any  other  protein  is  boiled  with  strong  acid  or 
alkali,  a  decomposition  takes  place  with  the  production  of  simpler 
substances.  These  simple  substances  resulting  from  such  decomposi- 
tion have  of  late  years  been  studied  by  a  number  of  investigators.  A 
similar  decomposition  occurs  when  the  protein  is  acted  upon  by 
proteolytic  enzymes.  These  enzymes  also  have  the  power  of  trans- 
forming casein  into  bodies  of  less  molecular  complexity.  The 
changes  are  of  a  hydrolytic  nature,  the  original  molecule  being 
broken  successively  at  different  places  and  a  molecule  of  water 
entering  at  the  point  of  cleavage.  The  extent  to  which  the  protein 
is  hydrolyzed,  and  consequently  the  nature  of  the  resulting  products, 
depends  upon  the  enzyme.  Pepsin,  obtained  from  the  gastric  juice, 
does  not  carry  the  proteolysis  beyond  the  peptone  stage,  while  tryp- 


NITROGENOUS   CONSTITUENTS   OF   CHEESE.  11 

sin,  obtained  from  the  pancreas,  breaks  up  the  protein  into  crystal- 
line end  products.  Erepsin,  on  the  other  hand,  does  not  attack 
native  proteins,  with  the  exception  of  casein,  but  acts  readily  upon 
proteoses  and  peptones.  The  enzyme  isolated  by  the  writer  from 
Camembert  mold  resembles  erepsin  in  this  respect. 

NITROGENOUS  CONSTITUENTS  OF  THE  CHEESE. 

The  ripening  of  Camembert  cheese  being  a  proteolysis,  certain 
digestion  products  may  be  expected  to  occur  in  the  ripened  cheese. 
As  casein,  or  rather  paracasein,  forms  the  main  bulk  of  the  proteins 
in  the  curd,  it  would  therefore  undergo  the  same  changes  that  occur 
when  casein  is  digested  artificially  with  an  enzyme,  though  the 
proteolysis  is  never  allowed  to  go  en  as  far  in  a  cheese  as  is  usually 
done  in  an  artificial  digestion  experiment.  The  products  may  be 
grouped  roughly  into  the  following  classes:  Caseoses,  peptones, 
polypeptids,  amino-acids,  and  ammonia.  Methods  for  the  separation 
of  these  groups  of  substances  from  cheese  have  already  been  elabo- 
rated by  Van  Slyke  and  Hart.11  They  consist  in  extracting  the  cheese 
with  water,  and  determining  the  nitrogen  in  the  precipitates  obtained 
by  the  addition  of  various  reagents  to  aliquot  portions  of  this  extract. 

In  the  subsequent  pages  of  this  article  the  separation  of  the  indi- 
vidual members  of  these  groups  will  be  discussed.  The  cheeses  used 
for  this  work  were  made  at  the  Storrs  Experiment  Station,  and  were 
pronounced  by  experts  to  be  equal  in  texture  and  appearance  to  the 
imported  brands.  Both  texture  and  flavor  showed  them  to  be  excel- 
lent cheeses  of  the  Camembert  type. 

The  analysis  of  a  ripe  cheese  by  the  method  of  Van  Slyke  and  Hart 
showed  the  nitrogenous  constituents  to  be  present  in  the  following 
amounts : 

Per  cent 
N  itrogen  as —  of  cheese. 

Total  nitrogen 2. 47 

Water— soluble « 1 .  79 

Precipitated  by  hydrochloric  acid 40 

Caseoses 10 

Peptones 

Amino-acids 82 

Salt — soluble  (paracasein) 15 

Ammonia 19 

The  polypeptids  are  included  under  the  peptones  and  amino-acids. 
This  represents  the  analysis  of  an  individual  cheese.  Some  varia- 
tions occur  with  different  samples,  but  the  differences  are  only  slight 
with  cheeses  at  the  same  stage  of  ripening.  As  none  of  the  individual 
members  of  these  groups  of  digestion  products  had  ever  been  isolated 
from  Camembert  cheese,  a  determination  of  some  of  the  more  char- 
acteristic ones  was  considered  advisable.  Proximate  analyses  of  the 


12  CHANGES   IN    RIPENING   OF   CAMEMBERT   CHEESE. 

cheese  at  different  stages  of  ripening  will  be  found  in  Bosworth's 
paper.15  These  analyses  show  merely  the  rate  of  formation  or  destruc- 
tion of  three  broad  groups  of  digestion  products,  and  throw  little 
light  upon  the  nature  of  the  ripening  from  the  biochemical  stand- 
point. The  writer  has  attempted  to  determine,  as  far  as  the  facilities 
at  his  command  permitted,  the  relative  amounts  of  the  more  impor- 
tant members  occurring  in  these  groups.  They  will  be  discussed  in 
the  same  order  as  the  groups  are  determined  in  Van  Slyke  and 
Hart's  method  for  the  analysis  of  cheese. 

HYDROCHLORIC    ACID    PRECIPITATE. 

When  an  aqueous  extract  of  the  cheese  is  made  and  acidified  with 
hydrochloric  acid,  a  white  curdy  precipitate  appears,  which  on  warm- 
ing to  50°  C.  clots  together  into  a  gummy  mass.  If  the  digestion 
were  of  a  peptic  nature  this  precipitate  should  consist  of  paranuclein. 
The  chief  characteristics  of  this  altered  phosphoprotein  are  its  high 
phosphorus  content  and  the  comparative  slowness  with  which  it  is 
further  acted  upon  by  pepsin.  On  the  other  hand,  trypsin  converts 
casein  directly  into  caseoses  without  the  formation  of  paranuclein, 
and  at  the  same  time  liberates  the  phosphorus  in  the  form  of  phos- 
phoric acid.  Erepsin  probably  acts  in  the  same  way  as  trypsin  in  this 
respect.  Again,  the  precipitate  might  be  a  coagulose  or  protein  syn- 
thesized by  the  reversed  action  of  a  proteolytic  enzyme.  Such  a 
coagulose  is  Kurajeff  s  plastein,  formed  by  the  action  of  rennet  on 
albuminoses. 

A  similar  precipitate  has  been  obtained  from  other  varieties  of 
cheese  and  has  usually  been  regarded  as  paranuclein.  As  the  ripen- 
ing of  Camembert  cheese  is  not  a  peptic  digestion,  it  seemed  unlikely 
that  this  precipitate  could  be  paranuclein.  In  order  to  determine 
more  precisely  the  nature  of  the  precipitate,  several  cheeses  were 
subjected  to  the  following  treatment. 

An  aqueous  extract  was  made  by  stirring  the  macerated  cheese 
with  water  in  a  bath  maintained  at  a  temperature  of  50°  C.  This 
extraction  was  repeated  several  times,  filtering  off  the  liquid  at  the 
end  of  half  an  hour  through  cotton  and  asbestos,  and  adding  fresh 
quantities  of  water  until  the  filtrate  was  practically  free  from  nitroge- 
nous matter.  The  extract  made  in  this  way  was  acidified  to  0.2  per 
cent  with  hydrochloric  acid,  whereupon  a  white  curdy  precipitate 
resulted.  The  precipitate  was  washed  thoroughly  with  acidulated, 
then  with  distilled,  water,  and  finally  dried  in  a  desiccator  and 
extracted  with  ether  to  remove  adhering  fat. 

Upon  testing  the  solubilities  of  this  precipitate,  it  was  found  that 
a  small  part  of  it  dissolved  in  5  per  cent  sodium  chlorid  solution, 
while  the  greater  part  was  soluble  in  50  per  cent  alcohol.  The 
smaller  fraction  was  studied  first.  It  dissolved  in  alkalies,  was 
reprecipitated  by  acids,  excess  of  which  dissolved  the  precipitate. 


HYDROCHLORIC    ACID    PRECIPITATE. 


13 


The  substance  was  readily  attacked  by  trypsin,  dissolving  com- 
pletely in  twenty-four  hours,  and  giving  a  solution  from  which  no 
precipitate  was  obtained  by  saturation  with  ammonium  sulphate. 
A  sample  dried  at  110°C.  gave  the  following  analysis,  based  upon 
the  ash-free  substance.  Alongside  it  are  given  Rose's  analysis  of 
paracasein  and  Chittenden's 1S  analysis  of  paranuclein,  with  the 
phosphorus  as  found  by  Jackson. 


Substance 
from  cheese. 

Paracasein 
(Rose). 

Paranuclein 
(Chittenden). 

Carbon        .. 

53.63 

53  94 

51  29 

Hydrogen 

7  08 

7  14 

7  2C 

Nitrogen 

15  10 

15  14 

15  23 

Sulphur  

.98 

1  01 

08 

Phosphorus 

50 

2  75 

Ash                      

82 



12  43 

A  comparison  of  these  analyses  shows  that  the  substance  is  too 
low  in  phosphorus  to  be  paranuclein.  The  analysis  agrees  fairly 
well  with  that  of  paracasein,  and  it  can  be  regarded  as  paracasein 
from  which  a  part  of  the  phosphorus  has  been  liberated  by  enzyme 
action.  Paracasein  probably  contains  about  the  same  amount  of 
phosphorus  as  casein  itself,  which,  according  to  Hammarstein,  is 
0.85  per  cent. 

The  alcohol-soluble  part  of  the  precipitate  was  dissolved  in  50  per 
cent  alcohol,  filtered  and  poured  into  water.  A  gummy  precipitate 
was  formed.  It  was  dried  at  110°  C.,  extracted  with  ether,  and 
analyzed.  On  fusing  the  substance  with  potash  and  niter  for  the 
sulphur  determination,  a  strong  odor  of  skatol  was  emitted,  stronger 
than  that  obtained  with  casein.  This  indicates  the  presence  of  the 
tryptophane  group.  The  ready  solubility  in  alcohol  and  insolu- 
bility in  water  are  properties  characteristic  of  caseoglutin,  a  sub- 
stance discovered  in  Swiss  cheese  by  Weidmann.  The  analysis, 
together  with  Rose's  analysis  of  caseoglutin,  follows: 


Substance 
from  cheese. 

Caseoglutin 
(Rose). 

Carbon                                    

54.34 

54.4 

7.30 

7.34 

Nitrogen                        .          

15.37 

15.29 

.95 

.95 

.06 

\sh 

.28 

Both  the  analysis  and  the  properties  of  tills  substance  agree  with 
those  of  caseoglutin. 


COAOULABLK    PKOTKINS. 


Winterstein  found  in  Swiss  cheese  a  small  amount  of  a  substance 
which  was  precipitated  from  acid  solution  by  boiling.  This  substance 
he  calls  tyroalbumin.  Its  exact  nature  has  not  yet  been  determined. 


14  CHANGES  IN    RIPENING   OF   CAMEMBERT   CHEESE. 

Several  attempts  were  made  to  find  this  substance  in  Camembert 
<-hee>e.  but  so  far  they  have  resulted  in  failure.  On  heating  the 
filtrate  from  the  eascoglutin  to  boiling,  both  in  acid  and  in  neutral 
solution,  no  precipitate  was  obtained. 

CA8EOSES. 

These,  together  with  the  peptones,  are  the  intermediate  disintegra- 
tion of  casein  by  ordinary  proteolytic  enzymes.  They  can  not  be 
regarded  as  homogeneous  substances,  as  they  represent  transition 
products  formed  by  the  loss  of  varying  numbers  of  ammo-acid  mole- 
cules from  the  original  protein.  They  can,  however,  be  separated 
into  groups  according  to  their  solubilities.  A  method  for  the  separa- 
tion of  albumoses  by  fractional  precipitation  with  ammonium  sul- 
phate was  elaborated  by  Pick.19  If  nitrogen  determinations  are  to 
be  made,  the  ammonium  sulphate  must  be  removed  by  dialysis,  a 
long  and  tedious  operation.  To  obviate  this  difficulty  Zunz 20  used 
zinc  sulphate  and  found  that  the  precipitation  limits  were  quite  as 
sharply  defined.  As  the  elementary  analyses  of  these  groups  of 
caseoses  have  very  little  value,  and  the  peptones  were  to  be  separated 
from  the  filtrate,  the  method  of  Pick  was  used  in  this  work.  These 
saturation  limits  can  not,  however,  be  regarded  as  reliable  indexes  of 
individuality. 

The  caseoses  of  the  cheese  were  separated  into  the  four  fractions 
described  by  Pick.  They  are  designated  as  follows:  Protocaseose, 
by  half  saturation  of  the  neutral  solution  with  ammonium  sulphate; 
deuterocaseose  A,  by  two-thirds  saturation;  deuterocaseose  B,  by 
complete  saturation;  and  deuterocaseose  C,  by  acidifying  the  filtrate 
from  B.  In  the  early  stages  of  ripening,  the  protocaseose  predomi- 
nates. In  the  ripened  cheese,  however,  protocaseose  and  deutero  B 
are  present  in  about  equal  amounts,  and  together  form  about  three- 
fourths  of  all  the  caseoses.  A  distinction  will  be  noticed  here  from 
the  albumose  formation  observed  by  Zunz  in  peptic  digestion.  Ac- 
cording to  Zunz,  after  deutero  B  has  reached  its  maximum,  deutero 
A  predominates,  and  finally  deutero  C. 

In  purifying  the  different  fractions,  the  method  of  Haslam21  was 
followed  out,  viz,  rubbing  the  precipitate  in  a  mortar  with  am- 
monium sulphate  solution  of  the  same  concentration  as  the  filtrate. 
Several  reprecipitations  were  made  before  the  product  was  finally 
freed  from  ammonium  sulphate  by  repeated  precipitation  with  alcohol. 

The  first  fraction  should  contain,  besides  protocaseose,  heteroal- 
bumose  if  tliis  substance  were  present  in  the  cheese.  Heteroal- 
bumose  could  not  be  derived  from  casein.  Traces  were  found,  how- 
ever, but  they  probably  came  from  albumin.  Upon  subjecting  the 
carefully  purified  protocaseose  to  dialysis,  a  slight  residue  was  left 


PEPTONES   AND    POLYPEPTIDS.  15 

which  would  not  diffuse.  The  amount  was  too  small  for  chemical 
examination,  but  it  was  probably  heteroalbumose.  Ah1  the  fractions 
gave  the  biuret  reaction,  and  all  except  deutero  C  gave  the  Millon 
reaction.  The  intensity  of  the  lead  sulphid  reaction  seemed  to 
diminish  progressively,  until  with  deutero  C  it  was  just  perceptible. 

PEPTONES. 

•  The  filtrate  from  the  caseoses  was  nearly  neutralized  with  ammonia 
and  treated  with  a  saturated  solution  of  ferric  ammonium  sulphate. 
A  gelatinous  brown  precipitate  resulted.  This  corresponds  to  the 
alpha  and  beta  peptones  of  Siegfried. 22  The  precipitate  was  filtered 
off,  washed  with  a  saturated  solution  of  iron  alum,  and  decomposed 
by  barium  hydrate.  After  filtering  off  the  ferric  hydroxid  and  ba- 
rium sulphate,  a  current  of  air  was  drawn  through  the  alkaline  solu- 
tion until  the  ammonia  was  expelled.  The  barium  was  then  removed 
by  sulphuric  acid,  and  the  solution  concentrated  under  diminished 
pressure  and  poured  into  a  large  volume  of  alcohol.  A  precipitate 
was  obtained  which  is  analogous  to  Winter-stem's  alpha  peptone. 
The  filtrate  still  contained  peptone,  as  was  shown  by  an  intense  biuret 
reaction.  Further  addition  of  alcohol  gave  no  precipitate.  The  solu- 
tion must  therefore  contain  an  alcohol-soluble  peptone — Winterstein's 
beta  peptone.  It  was  reprecipitated,  after  distilling  off  the  alcohol, 
by  phosphotungstic  acid,  the  precipitate  decomposed  by  barium  hy- 
droxid, and  the  barium  removed  by  sulphuric  acid,  carefully  avoid- 
ing an  excess.  The  solution  was  then  evaporated  to  dryness.  The 
resulting  beta  peptone  may  have  contained  slight  admixtures  of  poly- 
peptids,  but  further  purification  was  not  attempted. 

The  alpha  peptone  gave  no  Millon  reaction  and  a  strong  furfurol 
reaction.  Beta  peptone,  on  the  other  hand,  gave  a  strong  Millon 
reaction,  but  no  furfurol  reaction.  Both  gave  the  characteristic  red 
biuret  reaction,  xanthoproteic  reaction,  and  a  slight  lead  sulphid  reac- 
tion. Dried  at  105°  C.,  alpha  gave  15.10  and  beta  14.80  per  cent 
nitrogen.  In  this  case  the  analytical  figures  have  very  little  value, 
as  the  substances  are  hygroscopic,  and  on  drying  continue  to  lose 
water  until  a  temperature  is  reached  which  causes  a  slight  decompo- 
sition. The  two  peptones  were  present  in  about  equal  amount  and 
together  comprised  about  1.6  per  cent  of  the  cheese.  They  had  the 
bitter  taste  characteristic  of  peptones. 

POLYPEPTIDS. 

After  removal  of  the  caseoses  and  peptones  by  means  of  lead  acetate, 
carefully  avoiding  an  excess  of  the  reagent,  the  cheese  extract  still 
gave  a  biuret  reaction.  The  polypeptids  are  the  intermediate  prod- 
ucts between  peptones  and  amino-acids.  Some  of  them  give  a  biuret 


16  CHANGES   IN    RIPENING   OF   CAMEMBERT   CHEESE. 

reaction,  and  their  presence  is  probably  the  explanation  of  this  phe- 
nomenon. Some  are  precipitated  by  lead  acetate,  while  others  re- 
main in  solution.  Fischer  and  Abderhalden  obtained  by  the  tryptic 
digestion  of  casein  a  polypeptid,  which  on  hydrolysis  yielded  alpha- 
pyrrolidin-carboxylic  acid  and  phenylalanin.  These  two  acids  were 
not  found  in  the  free  state,  and  their  absence  has  b^en  regarded  as 
characteristic  of  tryptic  digestion.  Whether  or  not  erepsin  decom- 
poses this  polypeptid  is  not  yet  known.  It  is  very  probable  that  other 
polypeptids  exist,  temporarily  at  least,  as  transition  products  from 
the  peptones  to  the  amino-acids.  Many  of  them  would  be  destroyed 
by  the  action  of  the  mold,  while  others  would  be  more  resistant. 
Abderhalden23  found  that  Aspergillus  niger  grows  readily  on  glycyl- 
glycin  and  dileucylglycylglycin,  two  polypeptids  that  are  not  attacked 
by  trypsin.  Winterstein  regards  his  alpha  peptone  as  similar  in  many 
respects  to  Fischer's  polypeptid.  This,  however,  would  seem  im- 
probable, in  view  of  the  fact  that  he  has  demonstrated  phenylalanin 
and  prolin  in  the  cheese.  No  satisfactory  method  has  yet  been  found 
for  separating  the  polypeptids  as  a  group,  and  the  amount  present  in 
a  cheese  can  only  be  a  matter  of  conjecture.  The  polypeptids  will 
be  made  the  subject  of  future  study  in  this  connection. 

DIAMINOACIDS. 

The  next  group  of  substances  to  be  studied  was  the  diamino-acids, 
or  hexone  bases.  The  three  hexone  bases,  along  with  ammonia,  can 
be  determined  quantitatively,  and  for  that  reason  they  have  received 
more  attention  from  investigators  studying  the  disintegration  prod- 
ucts of  the  proteins  than  have  the  monoamino-acids.  They  can  be 
expressed  in  definite  figures,  whereas  the  other  disintegration  products 
have  to  be  expressed  in  minimal  values.  They  have  already  been 
found  in  cheese — in  Swiss  cheese  by  Winterstein  and  ThOny,  and  in 
Cheddar  cheese  by  Van  Slyke  and  Hart.  Owing  to  the  softer  con- 
sistency of  Camembert  cheese  a  somewhat  different  method  of  pro- 
cedure was  adopted  in  making  the  extraction. 

Three  kilograms  of  the  thoroughly  ripened  cheese  made  at  the 
Storrs  Experiment  Station  were  ground  in  a  mortar  and  extracted  six 
times  with  warm  water,  according  to  the  usual  method  of  analysis, 
until  the  volume  of  the  liquid  was  about  six  liters.  The  greater  part 
of  the  fat  rose  to  the  surface  and  could  easily  be  skimmed  off.  It 
was  washed  by  stirring  thoroughly  with  cold  water  and  the  filtrate 
mixed  with  the  cheese  extract.  The  remainder  of  the  fat  was  found 
to  be  precipitated  almost  quantitatively  with  the  proteins,  and  thus 
the  necessity  of  extracting  the  original  cheese  with  ether  was  obviated. 
The  extract  was  filtered  through  cotton  and  through  asbestos,  then 
acidified  with  sulphuric  acid  and  warmed  until  the  caseoglutin  had 


DIAMINO-ACIDS.  17 

settled  out,  whereupon  the  liquid  was  filtered  again.  The  solution 
was  now  concentrated  at  a  low  temperature  until  the  volume  was 
about  two  liters.  About  three  volumes  of  alcohol  were  added  to 
precipitate  the  greater  part  of  the  caseoses  and  peptones.  After 
filtering  off  this  precipitate  the  alcohol  was  distilled  off  under  dimin- 
ished pressure,  and  tannic  acid  added  to  precipitate  the  rest  of  the 
caseoses  and  peptones.  The  excess  of  tannic  acid  was  removed  by 
lead  acetate  and  the  lead  by  sulphuric  acid.  The  resulting  solution 
still  gave  a  biuret  reaction.  It  contained,  besides  traces  of  second- 
ary disintegration  products  and  polypeptids,  the  hexone  bases, 
ammo-acids,  and  ammonium  salts,  together  with  the  sodium  chlorid 
present  in  the  cheese. 

The  hexone  bases  were  precipitated  by  a  50  per  cent  solution  of 
phosphotungstic  acid  in  the  presence  of  5  per  cent  sulphuric  acid.  A 
large  amount  of  this  reagent  had  to  be  added  before  the  precipitation 
was  complete,  and  a  voluminous  white  precipitate  was  obtained. 
After  standing  several  days  it  was  filtered  off  and  washed  writh  5  per 
cent  sulphuric  acid  containing  a  little  phosphotungstic  acid.  The 
washing  was  a  long  and  tedious  operation.  It  was  found  necessary 
to  remove  the  precipitate  each  time  from  the  funnel  and  grind  it  with 
the  sulphuric  acid  in  a  mortar.  This  was  repeated  until  all  of  the 
sodium  salts  had  been  removed. 

For  the  separation  of  the  bases  KossePs24  older  method  was  used, 
after  removal  of  the  phosphotungstic  and  sulphuric  acids  by  barium 
hydroxid  and  passing  in  a  current  of  air  to  expel  the  ammonia.  The 
excess  of  barium  was  removed  by  carbon  dioxid,  and  mercuric 
chlorid  added  to  precipitate  the  histidin.  This  precipitate  was 
allowed  to  stand  several  days,  then  filtered  and  washed  again.  It 
was  suspended  in  water  and  decomposed  by  hydrogen  sulphid  after 
slightly  acidifying  with  sulphuric  acid.  The  filtrate  from  the  mer- 
curic sulphid  was  boiled  with  charcoal  until  practically  colorless, 
and  precipitated  with  silver  nitrate  and  ammonia.  The  arginin  was 
precipitated  by  saturating  the  solution  with  barium  hydroxid,  and 
adding  silver  nitrate  until  a  drop  of  the  solution  gave  a  brown  color 
on  the  addition  of  silver  nitrate.  The  arginin  silver  was  decomposed 
by  hydrochloric  acid  and  hydrogen  sulphid,  filtered,  boiled  with 
charcoal,  and  evaporated  to  crystallization.  The  filtrate  from  the 
arginin  was  freed  from  barium  and  silver  by  means  of  sulphuric  acid 
and  hydrogen  sulphid,  and  an  alcoholic  solution  of  picric  acid  added. 
The  lysin  picrate  did  not  crystallize  readily,  but  after  several  crystal- 
lizations the  characteristic  yellow  needles  were  obtained.  The  bases 
were  found  in  the  following  amounts:  IILstidin,  1.1  grains;  arginin, 
0.6  gram;  and  lysin,  1.9  grains.  Histidin  was  analyzed  in  the  form 
of  the  silver  salt,  arginin  as  the  chlorid,  and  lysin  as  the  picrate.  The 


(   HAXCKS    IX    RIPKXIXc;    OF    CAMKMBERT    CHEESE. 


free  histidin  gave  an  intense  red  color  with  diazobenzenesulphanilic 
acid.     The  analyses  of  the  bases  are  given  below: 

Histidin  silver,  C^HjN3O2Ag2II2O. 

( Beta-imidoazol-alpha-aminopropionlc  add.) 


Argon  turn. 
Nitrogen.. 


Calculated. 


V..M 
10.85 


Found. 


55.  00 
10.78 


Arginin  hydrochlorid,  C6Hl4N4O.2ltCl. 

( Delta-guanidinc-alpha-aminovalerianic  acid. ) 


Chlorin.. 
Nitrogen. 


Calculated. 


16.87 
22.  GO 


Found. 


Hi.  70 
22.50 


Lynn  picrate,C<HuN202CJf3N307. 
(Diaminocaproic  acid.) 


Calculated. 

Found. 

Nitrogen 

18.  00 

18.50 

Carlx>n 

38.40 

38.53 

Hydrogen 

4.53 

4.54 

The  other  bases  were  present  in  so  small  amount  (about  0.5  gram) 
that  no  attempt  was  made  to  isolate  them.  A  noteworthy  fact  is  that 
arginin,  which  was  not  found  at  all  in  Swiss  cheese,  is  present  here. 
It  is  possible  that  some  of  it  is  further  hydrolyzed  into  guanidin  and 
aminovalerianic  acid  or  into  urea  and  ornithin  (diaminovalerianic 
acid).  The  filtrate  from  the  histidin,  arginin,  and  lysin  had  a  very 
faint  but  characteristic  odor  of  tetramethylenediamin.  This  sub- 
stance would  result  from  the  liberation  of  carbon  dioxid  from 
ornithin,  one  of  the  cleavage  products  of  arginin.  It  could  not  have 
been  present,  however,  in  more  than  traces.  An  attempt  was  made 
to  separate  guanidin  in  the  form  of  the  gold  salt,  but  no  crystals  could 
be  obtained.  The  small  amount  of  bases  remaining  in  the  lysin  frac- 
tion indicates  that  the  occurrence  of  secondary  reactions  in  this  group 
is  very  slight. 

MONOAMINO-ACIDS. ' 

A  complete  separation  of  all  the  amino-acids  remaining  in  solution 
after  removal  of  the  intermediate  disintegration  products  and  hexone 
bases  can  be  accomplished  only  by  Fischer's  method  of  distilling  the 
ethyl  esters  in  vacuo.  This  necessitates  delicate  and  costly  apparatus 


MONOAMINO-ACIDS. 


19 


which  the  writer  did  not  have  at  his  command.  Certain  of  the  amino- 
acids,  however,  can  be  separated  almost  quantitatively  from  the  mix- 
ture without  resorting  to  the  method  of  esterification.  Among  these 
are  glutaminic  acid,  tyrosin,  and  leucin. 

Another  lot  of  cheese  (3  kilograms)  was  treated  in  the  manner 
described  above  to  remove  the  caseoglutin,  caseoses,  and  peptones. 
The  residue  was  evaporated  to  a  small  bulk,  saturated  with  hydro- 
chloric-acid gas,  and  kept  at  zero  for  several  days.  Crystals  were 
deposited  on  the  walls  of  the  flask,  and  a  pulverulent  precipitate  sepa- 
rated out  on  the  bottom.  These  were  found  to  consist  of  glutaminic 
acid,  hydrochlorid,  and  sodium  chlorid.  They  were  transferred  to  a 
Buchner  funnel  and  washed  with  concentrated  hydrochloric  acid, 
then  dissolved  in  water.  The  solution  was  neutralized  with  caustic 
soda  and  boiled  with  freshly  precipitated  copper  hydroxid.  A  blue 
precipitate  was  formed.  It  was  filtered  and  washed,  and  then  sus- 
pended in  water  slightly  acidified,  and  decomposed  by  hydrogen  sul- 
phid.  The  free  acid  thus  obtained  was  again  saturated  with  hydro- 
chloric-acid gas  and  allowed  to  crystallize  as  before.  The  colorless 
crystals  thus  obtained  were  decomposed  by  the  calculated  amount  of 
caustic  soda  (30  c.  c.  N-NaOH),  and  the  free  acid  crystallized  out. 
About  5  grams  of  crystals  were  obtained.  Analysis  gave  the  follow- 
ing figures : 

(Hutaminic  acid,  C5H9A:Ot. 
(Aminoglutaric  acid.) 


Calculated. 

Found. 

Carbon 

40.82 

40.  62 

Hydrogen  .  .  . 

6.13 

fi.  15 

Nitrogen 

9.52 

9.53 

The  filtrate  from  the  glutaminic  acid  was  treated  with  lead  carbon- 
ate to  remove  the  bulk  of  the  hydrochloric  acid,  and  the  lead  remain- 
ing in  solution  was  removed  by  sulphuric  acid.  After  filtering  and 
neutralizing,  the  solution  was  evaporated  to  incipient  crystallization. 
The  first  crop  of  crystals  should  contain  tyrosin  and  traces  of  leucin, 
and  the  second  leucin  with  traces  of  tyrosin.  The  two  constituents 
of  each  fraction  were  separated  by  treatment  with  glacial  acetic  acid. 
The  leucin  purified  in  tliis  way  gave  no  color  with  Millon's  reagent. 
The  tyrosin  was  tested  for  sulphur  by  fusing  a  portion  of  it  with 
sodium  carbonate  and  adding  sodium  nitroprussid  to  the  aqueous 
solution.  No  coloration  was  obtained,  indicating  the  absence  of 
cystin.  About  8  grams  of  tyrosin  and  14  grams  of  loucin  were 
obtained.  It  must  be  borne  in  mind  that  while  the  greater  part  of  the 
glutaminic  acid  and  leucin  can  be  isolated  in  this  way,  the  amounts 
do  not  represent  strictly  quantitative  results,  for  a  further  yield  is 


20 


CHANGES   IN    RIPENING   OF    CAMEMBEKT   CHEESE. 


invariably  obtained  from  the  higher  boiling  fractions  of  the  ethyl 
esters.     Following  are  the  analyses  of  the  tyrosin  and  leucin: 

Tyrosin,  C9Hn^O^. 
(P-hydroxyphenyl-alpha-amlnopropionlc  acid.) 


Calculated. 

Found. 

Carbon  

59.66 

59.53 

Hydrogen  

6.07 

(i.OO 

Nitrogen  

7.73 

Leucin,  C6//13A7O2. 
(Alpha-aminoisobutylacetic  acidJ 


Calculated. 

53.96 
9.92 
10.68 

Found. 

First 
crop. 

Second 
crop. 

Carlwn  

54.99 
10.53 

54.83 

9.62 
10.76 

It  v<lrogen  

Nitrogen 

The  tyrosin  gave  the  characteristic  color  reactions,  viz,  a  red  color 
and  precipitate  with  Millons  reagent,  a  bright  red  coloration  with 
diazobenzenesulphanilic  acid,  and  a  yellow  precipitate  of  nitro ty- 
rosin nitrate  with  nitric  acid.  Under  the  microscope  it  showed  the 
characteristic  wavy  needles. 

The  leucin  was  also  characteristic  under  the  microscope.  Heated 
on  a  platinum  foil  it  sublimed  completely,  emitting  an  odor  like  that 
of  some  of  the  higher  alkylamins. 

On  further  concentrating  the  filtrate  from  the  leucin,  a  few  needle- 
shaped  crystals  were  obtained  having  a  sour  taste.  They  did  not 
melt  at  225°  C.  Not  enough  of  the  substance  was  obtained  for 
analysis,  but  it  was  probably  aspartic  acid. 

As  has  already  been  mentioned,  the  remainder  of  the  amino- 
acids  can  not  be  separated  without  resorting  to  Fischer's  method  of 
distillation.  Phenylalanin  and  tryptophan,  however,  can  be  detected 
qualitatively.  On  evaporating  the  solution  to  dryness  and  treating 
the  residue  with  sulphuric  acid  and  potassium  dichromate,  the 
writer  felt  confident  that  he  detected  the  odor  of  phenylacetaldehyde, 
notwithstanding  the  presence  of  other  aldehydes  formed  by  the  oxida- 
tion with  dichromate.  This  would  indicate  the  presence  of  phenyl- 
alanin,  but  from  this  test  alone  it  would  be  impossible  to  say  with 
certainty  whether  phenylalanin  was  really  present  or  not. 

A  striking  fact  is  that  none  of  the  cheeses  examined  responded  to 
the  tryptophan  reaction  with  acetic  acid  and  bromin  water.  Aque- 


ABSENCE  OF  PUTREFACTIVE  PRODUCTS.  21 

ous  extracts  of  cheeses  in  all  stages  of  ripening  were  examined,  but 
in  all  cases  they  failed  to  give  any  coloration  with  this  reagent. 
When  the  mold  was  grown  upon  milk  the  tryptophan  reaction  was 
likewise  negative,  although  the  casein  was  broken  down  into  amino- 
acids,  among  which  leucin  and  tyrosin  were  identified.  On  the  other 
hand,  the  enzyme  preparation  from  the  same  mold  readily  digests 
milk  in  the  presence  of  toluol,  and  the  tryptophan  reaction  is  inva- 
riably positive.  If  tryptophan  were  liberated  in  the  cheese,  it  might 
undergo  further  decomposition  as  a  result  of  bacterial  action.  In 
this  case  the  end  products  would  be  indol  and  skatol — characteristic 
products  of  putrefaction.  Both  of  these  substances  would  have 
given  a  coloration  with  the  tryptophan  reagent,  and  must  therefore 
have  been  absent. 

AMMONIA. 

X 

A  normal  cheese  contains  from  0.20  to  0.25  per  cent  of  ammonia. 
The  greater  part  of  this  is  in  combination  with  acid  radicals,  for  while 
the  ripened  cheese  is  alkaline  to  litmus  it  is  still  acid  to  phenolphtha- 
lein.  Some  of  the  more  highly  flavored  specimens,  however,  have  a 
distinct  odor  of  ammonia  near  the  rind.  The  formation  of  ammonia 
does  not  begin  until  after  the  second  week,  then  the  amount  steadily 
increases  until  the  cheese  is  ripe.  As  long  as  the  ripened  portion 
does  not  extend  more  than  a  few  millimeters  below  the  mycelium  of 
the  mold  no  ammonia,  either  free  or  in  combination,  can  be  detected. 
Cheeses  with  a  very  strong  flavor  contain,  as  a  rule,  more  ammonia 
than  the  milder  ones.  In  cheeses  that  are  overripe  the  presence  of 
free  ammonia  can  usually  be  detected  by  its  odor.  For  the  deter- 
mination of  ammonia,  the  writer  has  found  Folin's  method  more  sat- 
isfactory than  the  regular  method  of  distilling  the  tannin  filtrate  with 
magnesium  oxid.  It  can  be  used  in  the  presence  of  proteins  without 
effecting  any  decomposition. 

ABSENCE  OF  PUTREFACTIVE  PRODUCTS. 

While  the  ripened  cheese  possesses  a  peculiar  odor,  which  to  some 
people  is  quite  disagreeable,  it  does  not  resemble  that  of  the  typical 
putrefactive  products,  nearly  all  of  which  are  characterized  by  a  foul 
odor.  Among  the  substances  belonging  to  this  class  are  indol,  skatol, 
mercaptan,  hydrogen  sulphid,  and  phenols.  Qualitative  tests  were 
made  repeatedly  for  all  of  these  substances  on  different  samples  of 
cheese,  but  in  all  cases  they  were  negative,  except  in  those  cheeses 
that  had  gone  past  the  usual  ripening.  After  the  ripening  is  complete 
putrefaction  may  set  in  if  the  cheese  does  not  receive  proper  care.  In 
those  cases  where  some  of  the  putrefactive  products  were  found  the 
cheeses  were  otherwise  unfit  for  eating,  as  was  evidenced  by  a  very 


22  CHANGES   IN   RIPENING   OF   CAMEMBERT   CHEESE. 

disagreeable  odor  and  taste.  Nearly  all  of  these  *ubstanc?s  are 
found  in  Limburg  cheese,  but  they  do  not  occur  in  good  Camembert 
cheese.  The  failure  to  find  typical  putrefactive  products,  together 
with  the  fact  that  the  lysin  fraction  of  the  diamino-acids  contained 
only  traces  of  diamins,  indicates  that  whatever  the  action  of  the 
bacteria  in  the  cheese  may  be,  they  do  not  cause  secondary  reactions 
of  this  nature  to  any  extent. 

SUMMARY. 

The  following  substances  have  been  isolated  from  Camembert 
cheese:  Caseoglutin,  protocaseose,  deuterocaseoses  A,  B,  and  C,  alpha 
and  beta  peptones,  histidin,  arginin,  lysin,  glutarninic  acid,  tyrosin, 
and  leuein. 

Among  those  substances  which  the  writer  failed  to  find  are  para- 
nuclein,  tryptophan,  indol,  skatol,  mercaptan,  hydrogen  sulphid,  and 
phenols. 

The  ripening  of  Cam?mbert  cheese  can  not  be  a  peptic  digestion,  as 
is  shown  by  the  following  facts: 

1.  Paranuclein,  the  characteristic  product  of  peptic  digestion  of 
casein,  is  absent. 

2.  The  greater  part  of  the  phosphorus  is  liberated  and  appears  as 
acid    calcium   phosphate.     According    to    Plimmer    and    Bayliss,25 
pepsin  acts  slowly  and  incompletely,  only  70  per  cent  of  the  phos- 
phorus being  liberated  from  casein  in  149  days.,  and  that  mostly  in 
the  organic  form. 

3.  Amino-acids  and  ammonia  are  present  in  considerable  amount. 
The   ripening  resembles  ereptic   digestion  in  many   respects,    as 

follows: 

1.  The  reaction  of  the  cheese  before  ripening,  i.  e.,  acidity  caused  by 
acid  phosphates,  is  most  favorable  to  the  activity  of  ereptase. 

2.  The  digestion  proceeds  beyond  the  peptone  stage,  with  the  for- 
mation of  amino-acids  and  ammonia. 

3.  A  separate  study  of  the  enzyme  from  the  Camembert  mold 
shows  that  this  enzyme  is  a  vegetable  ereptase. 

The  absence  of  tryptophan,  which  is  ordinarily  liberated  in  ereptic 
digestion,  is  striking. 

The  presence  of  caseoglutin,  the  remarkable  albumose-like  body,  is 
also  noteworthy.  This  substance  has  not,  to  the  writer's  knowledge, 
been  observed  in  digestions  with  pure  enzymes. 

ACKNOWLEDGMENTS. 

In  conclusion,  the  writer  acknowledges  his  indebtedness  to  Prof. 
L.  B.  Mendel,  of  Yale  University,  and  to  Dr.  B.  B.  Turner,  formerly  of 
the  Storrs  Experiment  Station,  for  many  helpful  suggestions  in  carry- 
ing out  this  work. 


REFERENCES  TO  LITERATURE. 

1.  CHAPTAL.     Observations  sur  les  caves  et  le  fromage  de  Roquefort.     Annales  de 

Chimie,  tome  4,  pp.  31-61.     Paris,  1790. 

2.  BLONDEAU,  CH.     Etude  chimique  du  fromage  de  Roquefort.     Annales  de  Chimie 

et  de  Physique,  4th  ser.,  t.  1,  pp.  208-231.     Paris,  1864. 

3.  BRASSIER.     Sur  les  modifications  que  le  fromage  subit  en  vieillissant.     Annales 

de  Chimie  et  de  Physique,  4th  ser.,  t.  5,  pp.  270-294.     Paris,  1865. 

4.  SIEBER,  NADINA.     Ueber  die  angebliche  umwandlung  des  eiweisses  in  fett  beim 

reifen  des  Roquefort-kase.  Journal  fur  praktische  Chemie,  neue  folge,  band 
21,  pp.  203-221.  Leipzig,  1880. 

5.  JACOBSTAHL,  H.     Versucheiiber  die  fettbildungbei  der  reifung  des  kases.     Archiv 

fur  gesammte  Physiologic,  band  54,  pp.  484-500.     Bonn,  1893. 

6.  VON  NAGELI,  C.,  and  LOEW,  O.     Ueber  die  fettbilding  bei  den  niederen  pilzen. 

Journal  fur  praktische  Chemie,  neue  folge,  band  21,  pp.  97-114.     Leipzig.  1880. 

7.  WEIDMANN,  U.     Untersuchungen  tiber  die  zusammensetzung  und  den  reifungs- 

prozess  des  Emmenthaler  kiises.  Landwirtschaftliche  Jahrbiicher.  band  11, 
pp.  587-612.  Berlin,  1882. 

8.  ROSE,  B.,  und  SCHULZE,  E.     Ueber  einige  bestandtheile  des  Emmenthaler  kiises. 

Die  landwirtschaftlichen  Versuchsstationen.  band  31,  pp.  115-137.  Berlin, 
1885. 

9.  WINTERSTEIN,  E.,  and  THONY,  J.     Beitriige  zur  kenntniss  der  bestandtheile  des 

Emmenthaler  kases.  Mitteilung  I.  Zeitschrift  fiir  physiologische  Chemie, 
band  36,  heft  1,  pp.  28-38.  Strassburg.  1902. 

WINTERSTEIN,  E.  Uber  einige  bestandteile  des  Emmentaler  kases.  Mitteilung 
II.  Zeitschrift  fiir  physiologische  Chemie,  band  41,  heft  6.  pp.  485-504.  Strass- 
burg, 1904. 

WINTERSTEIN,  E.,  und  BISSEGGER,  W.  Zur  kenntnis  der  bestandteile  des  Emmen- 
taler kases.  Versuche  zur  bestimmung  der  stickstoffhaltigen  kases-bestand- 
teile.  Mitteilung  III,  Zeitschrift  fiir  physiologische  Chemie,  band  47,  heft  1, 
pp.  28-57.  Strassburg,  1906. 

10.  BABCOCK,  S.  M.,  RI.SSELL,  H.  L.,  and  VIVIAN,  A.     Influence  of  rennet  on  cheese 

ripening.  Wisconsin  Agricultural  Experiment  Station.  17th  Annual  Report, 
1900,  pp.  102-122.  Madison,  1900. 

BABCOCK,  S.  M.,  and  RUSSELL,  H.  L.  Unorganized  ferments  of  milk:  A  new 
factor  in  the  ripening  of  cheese.  Wisconsin  Agricultural  Experiment  Station, 
14th  Annual  Report,  1897.  pp.  161-193.  Madison,  1897. 

11.  VAN  SLYKE,  L.  L.,  and  HART,  E.  B.     Methods  for  the  estimation  of  the  proteolytic 

compounds  contained  in  cheese  and  milk.  New  York  Agricultural  Experiment 
Station,  Bulletin  215.  Geneva.  1902. 

Some  of  the  compounds  present  in  American  Cheddar  cheese.  New  York  Agri- 
cultural Experiment  Station,  Bulletin  219.  Geneva,  1902. 

VAN  SLYKE,  L.  L.,  HARDING,  H.  A.,  and  HART,  E.  B.  Rennet-enzyme  as  a 
factor  in  cheese-ripening.  New  York  Agricultural  Experiment  Station,  Bul- 
letin 233.  Geneva,  1903. 

12.  CONN,  H.  W.,  THOM,  CHARLES,  BOSWORTH,  A.  W.,  STOCKING,  jr.,  W.  A.,  and 

ISSAJEFF,  T.  W.  The  Camembert  type  of  soft  cheese  in  the  United  States. 
U;  S.  Department  of  Agriculture,  Bureau  of  Animal  Industry.  Bulletin  71. 
Washington,  1905.  (Also  issued  as  Storrs  Agricultural  Experiment  Station 

Bulletin  35.     Storre,  Conn.,  1905.) 

23 


24  »   HANCIS    IN     KII'KMN<;    < >1     «  A  M  I.  M  111  K  I     QHBEBE. 

13.  TUOM.  i  H.MM.KS.     Fmmi  in  cheese  ripening:  Camembert  and  Roquefort.     I 

Department  of  Agriculture,  Bureau  of  Animal  Industry.  Bulletin  82.  Washing- 
ton. 190(5. 

14.  ISSAJEFK,  THEODORE  W.     Directions  f<»r  making  the  Gamembert  type  of  cheese. 

I".  S.  Department  of  Agriculture.  Bureau  of  Animal  Industry.  Bulletin  98. 
Washington.  1907.  (Also  issued  as  Storrs  Agricultural  Experiment  Station 
Bull. -mi  Hi.  Siorrs.  Conn..  1907.) 

15.  BOSWOKTII.   Ai.KitKD   W.     Chemical  studies  of  Camemberi    chee-e.      \e\v    York 

Agricultural  Exj>eriment  Station.  Technical  Bulletin  5.     Geneva.  1907. 

16.  ROOERS,  LORE  A.     The  relation  of  bacteria  to  the-  flavors  of  Cheddar  cheese. 

I".  S.  Department  of  Agriculture,  Bureau  of  Animal  Industry,  Bulletin  62. 
Washington.  1904. 

17.  VIN  i:s.  S.  II.     The  proteases  of  plants.     Annals  of  Botany,  vol.  19,  No.  74,  pp.  171- 

1S7.     London.  1905. 

18.  CHITTENOEN.  R.  H.     Caaeoses,  casein  dyspeptone  and  casein  peptone.     Studies 

from  the  chemical  laboratory  of  the  Sheffield  Scientific  School,  Yale  Uni- 
versity, v.  3,  pp.  69. 

19.  PICK.  ERXST  P.     Untersuchungen  iiber  die  proteinstoffe.     Ein  neues  verfahren 

/.ur  trennung  von  albumosen  und  peptonen.  Zeitschrift  fiir  physiologische 
Chemie.  band  24,  heft  3,  pp.  246-275.  Strassburg,  1897. 

20.  ZUNZ,  E.     Die  fractionirte  abscheidung  der  peptischen  verdauungungs-produkte 

mittelst  zinksulfat.  Zeitschrift  fiir  physiologische  Chemie.  band  27.  heft  :•;. 
pp.  219-249.  Strassburg,  1899. 

•_'l.  HASLAM.  II.  C.     The  separation  of  proteids.    Journal  of  Physiology',  vol.  32, 
No.  :5-4,  pp.  267-298.     Cambridge,  England,  1905. 

22.  SIEGFRIED.  M.     Ueberantipepton.     Mittheilungl.     Zeitschrift  fiir  physiologische 

Chemie.  band  27,  heft  4-5,  pp.  335-347.     Strassburg.  1899. 

Ueberantipepton.     Mittheilung  II.     Zeitschrift  fiir  physiologische  Chemie.  band 
35,  heft  2,  pp.  164-191.     Strassburg,  1902. 

23.  ABDERHALDEX.  EMIL.     Eiweissfctoffe.     Abbau  und  aufbau  der  eiweisskorber  im 

tierischen  und  pflanzlichen  organLsmus.  Lehrbuch  der  physiologischen 
Chemie,  p.  236.  Berlin,  1906. 

24.  KOSSEL    A.     Ueber  die  constitution  der  einfachsten  eiweissstoffe.     Zeitschrift 

fiir  physiologische  Chemie,  band  25,  heft  3-4,  pp.  165-189.     Strassburg,  1898. 

25.  PLIMMER,  R.  H.  ADERS,  and   BAYLISS,  \V.  M.     The  separation  of  phosphorus 

from  caseinogen  by  the  action  of  enzymes  and  alkali.  Journal  of  Physiology, 
vol.  33,  No.  t;.  pp.  439-461.  Cambridge,  England,  1906. 

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